The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.


Introduction
• R38 Be consistent.If you refer every time to a plant -bacteria interaction.It is maybe best to address the symbiosis as Medicago/Sinorhizobium.In the rest of your paper, you are talking of D.sansibarennsis/ O. dioscoreae.• R69.Why is it important to state that nitrogen fixation is ruled out in the Dioscorea/Orella symbiosis, is it not in the other leaf nodulating interactions?

Material & Methods
• R108: ½ MS vs R115 half strength Murashige and Skoog • R116 16h/8h routine, maybe use this abbreviation for the rest of the methods (R132, R151, R244) • R117: The injection in the bulbils was it at a specific location and depth or random?• R124-R130: Strange construction, wouldn't it be better to put the names of the different methods between brackets after each sentence?• R134 Could it be possible to state that you will use two surface sterilisation protocols?The bleach+ ethanol protocol and the PPM protocol • R143: you write what is present in MS medium, is that the same in R108, R115, R140? • R143 2% sucrose is that (w/v) like R215? • R153-R159: suggestion: would a table with all the Orella strains, with the specific characteristics not be a good addition?• R153-159: why did you use both tags GFP en mCherry?• R196 a verb is missing.Is this the triple A-staining mentioned in Figure 1? • R236 'Leaf length, width, area, and acumen length were determined from photographs…' was this used to calculate the leaf area • R240: which statistical analysis was used?• R269: which statistical analysis were used?

Results
• If you are not combining results and discussion, it's strange to see references in this part.
(R275) Could these references be integrated in the discussion or the methods?? • Table 1: Maybe add in the caption which effect you were researching and the contact time.
• R273-296: why were the amounts of bulbils and shoot tips not mentioned in the material and methods.• R298-R328.This is a mix of Methods (some of the methods explained here are not mentioned in methods), results and discussion.▪ mcherry?Is it necessary to put it in italics here cf.there results of the GFPstrain R277 • R392-R407: you mention 24 plants, while in the methods, you start with 25 plants.In addition you mention in the methods that 18 plants were aposymbiotic, while in the results you only mention 14. • R398 which method of inoculation could give a better option, this dripping option was also not a good option with the aposymbiotic bulbils?• R404 which statistical test did you use to test whether the results were significant.
• R405-R407: why do you refer first to B and C and then to A. •

Discussion
• R439 Not clear here, if leaf nodules in Psychotria develop when the bacteria is absent.
• R450 Psychotria kirkii is a synonym of P. punctata  S1: o why not mentioned in the methods (R152-159) o Maybe add in which experiment they were used?o Why was E.coli used?
• The data underlying the statistics are missing, can these be added as well?
Figure 3: o Caption refers to stem lengths in C, while the graph refers to shoot length o Caption refers to the total leaf area of individual plants tracked over a period of 30 days.What are the dots and what is the line?o C I would remove the abbreviations apo, control and sym if you work with a colour legend o C are the dots around the boxplots the values?Why are these not on one line per phenotype?These dots are not identified in the caption or in the legend.

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Figure S1: o Add white circles to make the letters readable and consistent.o S should be a small letter and the same font size as b.(maybe use arrows to visualise specific structures o I can't find s on the figure E • Figure S2: o Is it possible to use a bigger font size on the axis o Why are the values spread over the X-axis, should be one line per condition (same remark as figure 3) • Figure S3 o Is it possible to use a bigger font on the axes o Use the same layout as Figure S2 o Why are the values spread over the X-axis, should be one line per condition (same remark as figure 3) • Table • R341 how did you test the presence of bacteria?Molecularly?•Table1:omaybe add contact time and which effect you were researching in your caption o 10°, does that mean that 1 or none CFU were found?C and D, the mucus, trichomes and bacteria can they be visualised?▪ K and L, can the trichomes be visualised.
• Figure 1: o why no white circle around G en H? o why is the resolution different between G-H, I-J, M-N? o Caption:▪ what is triple A staining, not mentioned in the text ▪